RESUMEN
The repertoire of modifications to bile acids and related steroidal lipids by host and microbial metabolism remains incompletely characterized. To address this knowledge gap, we created a reusable resource of tandem mass spectrometry (MS/MS) spectra by filtering 1.2 billion publicly available MS/MS spectra for bile-acid-selective ion patterns. Thousands of modifications are distributed throughout animal and human bodies as well as microbial cultures. We employed this MS/MS library to identify polyamine bile amidates, prevalent in carnivores. They are present in humans, and their levels alter with a diet change from a Mediterranean to a typical American diet. This work highlights the existence of many more bile acid modifications than previously recognized and the value of leveraging public large-scale untargeted metabolomics data to discover metabolites. The availability of a modification-centric bile acid MS/MS library will inform future studies investigating bile acid roles in health and disease.
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Ácidos y Sales Biliares , Microbioma Gastrointestinal , Metabolómica , Espectrometría de Masas en Tándem , Animales , Humanos , Ácidos y Sales Biliares/química , Metabolómica/métodos , Poliaminas , Espectrometría de Masas en Tándem/métodos , Bases de Datos de Compuestos QuímicosRESUMEN
The rumen microbiome - a remarkable example of obligatory symbiosis with high ecological and social relevance.
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Microbiota , Rumen , Animales , SimbiosisRESUMEN
A major challenge of analyzing the compositional structure of microbiome data is identifying its potential origins. Here, we introduce fast expectation-maximization microbial source tracking (FEAST), a ready-to-use scalable framework that can simultaneously estimate the contribution of thousands of potential source environments in a timely manner, thereby helping unravel the origins of complex microbial communities ( https://github.com/cozygene/FEAST ). The information gained from FEAST may provide insight into quantifying contamination, tracking the formation of developing microbial communities, as well as distinguishing and characterizing bacteria-related health conditions.
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Bacterias/aislamiento & purificación , Microbiota , Adulto , Microbioma Gastrointestinal , Humanos , Lactante , Unidades de Cuidados IntensivosRESUMEN
The ubiquity of plasmids in all prokaryotic phyla and habitats and their ability to transfer between cells marks them as prominent constituents of prokaryotic genomes. Many plasmids are found in their host cell in multiple copies. This leads to an increased mutational supply of plasmid-encoded genes and genetically heterogeneous plasmid genomes. Nonetheless, the segregation of plasmid copies into daughter cells during cell division is considered to occur in the absence of selection on the plasmid alleles. We investigate the implications of random genetic drift of multicopy plasmids during cell division-termed here "segregational drift"-to plasmid evolution. Performing experimental evolution of low- and high-copy non-mobile plasmids in Escherichia coli, we find that the evolutionary rate of multicopy plasmids does not reflect the increased mutational supply expected according to their copy number. In addition, simulated evolution of multicopy plasmid alleles demonstrates that segregational drift leads to increased loss frequency and extended fixation time of plasmid mutations in comparison to haploid chromosomes. Furthermore, an examination of the experimentally evolved hosts reveals a significant impact of the plasmid type on the host chromosome evolution. Our study demonstrates that segregational drift of multicopy plasmids interferes with the retention and fixation of novel plasmid variants. Depending on the selection pressure on newly emerging variants, plasmid genomes may evolve slower than haploid chromosomes, regardless of their higher mutational supply. We suggest that plasmid copy number is an important determinant of plasmid evolvability due to the manifestation of segregational drift.
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Evolución Biológica , Flujo Genético , Modelos Genéticos , Plásmidos/genética , Cromosomas Bacterianos , Escherichia coli , Frecuencia de los GenesRESUMEN
Horizontal gene transfer via plasmids plays a pivotal role in microbial evolution. The forces that shape plasmidomes functionality and distribution in natural environments are insufficiently understood. Here, we present a comparative study of plasmidomes across adjacent microbial environments present in different individual rumen microbiomes. Our findings show that the rumen plasmidome displays enormous unknown functional potential currently unannotated in available databases. Nevertheless, this unknown functionality is conserved and shared with published rat gut plasmidome data. Moreover, the rumen plasmidome is highly diverse compared with the microbiome that hosts these plasmids, across both similar and different rumen habitats. Our analysis demonstrates that its structure is shaped more by stochasticity than selection. Nevertheless, the plasmidome is an active partner in its intricate relationship with the host microbiome with both interacting with and responding to their environment.
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Bacterias/genética , Microbiota/genética , Plásmidos/genética , Rumen/microbiología , Animales , Transferencia de Gen HorizontalRESUMEN
Given the highly dynamic and complex nature of the human gut microbial community, the ability to identify and predict time-dependent compositional patterns of microbes is crucial to our understanding of the structure and functions of this ecosystem. One factor that could affect such time-dependent patterns is microbial interactions, wherein community composition at a given time point affects the microbial composition at a later time point. However, the field has not yet settled on the degree of this effect. Specifically, it has been recently suggested that only a minority of taxa depend on the microbial composition in earlier times. To address the issue of identifying and predicting temporal microbial patterns we developed a new model, MTV-LMM (Microbial Temporal Variability Linear Mixed Model), a linear mixed model for the prediction of microbial community temporal dynamics. MTV-LMM can identify time-dependent microbes (i.e., microbes whose abundance can be predicted based on the previous microbial composition) in longitudinal studies, which can then be used to analyze the trajectory of the microbiome over time. We evaluated the performance of MTV-LMM on real and synthetic time series datasets, and found that MTV-LMM outperforms commonly used methods for microbiome time series modeling. Particularly, we demonstrate that the effect of the microbial composition in previous time points on the abundance of taxa at later time points is underestimated by a factor of at least 10 when applying previous approaches. Using MTV-LMM, we demonstrate that a considerable portion of the human gut microbiome, both in infants and adults, has a significant time-dependent component that can be predicted based on microbiome composition in earlier time points. This suggests that microbiome composition at a given time point is a major factor in defining future microbiome composition and that this phenomenon is considerably more common than previously reported for the human gut microbiome.
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Biología Computacional/métodos , Microbioma Gastrointestinal , Modelos Biológicos , Adulto , Bases de Datos Genéticas , Femenino , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/fisiología , Humanos , Lactante , Masculino , Factores de TiempoRESUMEN
The European seabass (Dicentrarchus labrax) is a teleost remarkably adapted to a wide range of water salinity, through osmoregulatory mechanisms, mainly operating in the gills and the intestine. As an important aquaculture species, its rearing in low-salinity conditions offers benefits for its inland culture. However, this demands a full comprehension of the European seabass osmoregulatory mechanisms and its response to acclimation protocols. The purpose of this study was to evaluate different acclimation protocols in terms of osmoregularity and stress response, following transferring of European seabass juveniles from seawater to freshwater. In addition, nutrient absorption was also examined since drinking rates are sensitive to salinity. The acclimation challenge was applied through three protocols: direct transfer (0â¯h) to freshwater, gradual transfer during 3â¯h and during 72â¯h. The short- (1â¯h after complete change to freshwater) and long-term effects (after 2â¯months) of each acclimation protocol were evaluated by assessing the expression of 1. The osmoregulatory genes: Na+/K+-ATPase α1, Na+/K+/2Cl- 1 co-transporter, aquaporins 1 and 3, and the cystic fibrosis transmembrane conductance regulator; 2. The heat shock protein 70 gene; 3. The peptide transporter genes corresponding to PepT1a, PepT1b and PepT2. The short-term acclimation response was pronounced in both gills and the intestine affecting stress-, osmoregulatory- and nutrient-related gene expression. Long-term effects were only evident in the intestine. Direct transfer in freshwater mainly induced a long-term stress response, while the short-term effect was more pronounced in the 3â¯h-transfer, potentially due to handling. Our results suggest that although the European seabass can withstand direct transfer to low-salinity conditions, a gradual transfer is recommended to prevent long-term stress effects.
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Aclimatación , Peces/fisiología , Expresión Génica , Mucosa Intestinal/metabolismo , Salinidad , Animales , Peces/genética , Agua Dulce , Nutrientes/metabolismo , Osmorregulación , Agua de MarRESUMEN
Motivation: Plasmids and other mobile elements are central contributors to microbial evolution and genome innovation. Recently, they have been found to have important roles in antibiotic resistance and in affecting production of metabolites used in industrial and agricultural applications. However, their characterization through deep sequencing remains challenging, in spite of rapid drops in cost and throughput increases for sequencing. Here, we attempt to ameliorate this situation by introducing a new circular element assembly algorithm, leveraging assembly graphs provided by a conventional de novo assembler and alignments of paired-end reads to assemble cyclic sequences likely to be plasmids, phages and other circular elements. Results: We introduce Recycler, the first tool that can extract complete circular contigs from sequence data of isolate microbial genomes, plasmidome and metagenome sequence data. We show that Recycler greatly increases the number of true plasmids recovered relative to other approaches while remaining highly accurate. We demonstrate this trend via simulations of plasmidomes, comparisons of predictions with reference data for isolate samples, and assessments of annotation accuracy on metagenome data. In addition, we provide validation by DNA amplification of 77 plasmids predicted by Recycler from the different sequenced samples in which Recycler showed mean accuracy of 89% across all data types-isolate, microbiome and plasmidome. Availability and Implementation: Recycler is available at http://github.com/Shamir-Lab/Recycler. Contact: imizrahi@bgu.ac.il. Supplementary information: Supplementary data are available at Bioinformatics online.
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Bacterias/genética , Genoma Bacteriano , Metagenoma , Plásmidos , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Algoritmos , Escherichia coli/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
Heterologous display of enzymes on microbial cell surfaces is an extremely desirable approach, since it enables the engineered microbe to interact directly with the plant wall extracellular polysaccharide matrix. In recent years, attempts have been made to endow noncellulolytic microbes with genetically engineered cellulolytic capabilities for improved hydrolysis of lignocellulosic biomass and for advanced probiotics. Thus far, however, owing to the hurdles encountered in secreting and assembling large, intricate complexes on the bacterial cell wall, only free cellulases or relatively simple cellulosome assemblies have been introduced into live bacteria. Here, we employed the "adaptor scaffoldin" strategy to compensate for the low levels of protein displayed on the bacterial cell surface. That strategy mimics natural elaborated cellulosome architectures, thus exploiting the exponential features of their Lego-like combinatorics. Using this approach, we produced several bacterial consortia of Lactobacillus plantarum, a potent gut microbe which provides a very robust genetic framework for lignocellulosic degradation. We successfully engineered surface display of large, fully active self-assembling cellulosomal complexes containing an unprecedented number of catalytic subunits all produced in vivo by the cell consortia. Our results demonstrate that the enzyme stability and performance of the cellulosomal machinery, which are superior to those seen with the equivalent secreted free enzyme system, and the high cellulase-to-xylanase ratios proved beneficial for efficient degradation of wheat straw.IMPORTANCE The multiple benefits of lactic acid bacteria are well established in health and industry. Here we present an approach designed to extensively increase the cell surface display of proteins via successive assembly of interactive components. Our findings present a stepping stone toward proficient engineering of Lactobacillus plantarum, a widespread, environmentally important bacterium and potent microbiome member, for improved degradation of lignocellulosic biomass and advanced probiotics.
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Membrana Celular/metabolismo , Celulasa/química , Celulasa/metabolismo , Celulosa/metabolismo , Celulosomas/metabolismo , Lactobacillus plantarum/metabolismo , Celulasa/genética , Microbioma GastrointestinalRESUMEN
Microbial communities are essential to the function of virtually all ecosystems and eukaryotes, including humans. However, it is still a major challenge to identify microbial cells active under natural conditions in complex systems. In this study, we developed a new method to identify and sort active microbes on the single-cell level in complex samples using stable isotope probing with heavy water (D2O) combined with Raman microspectroscopy. Incorporation of D2O-derived D into the biomass of autotrophic and heterotrophic bacteria and archaea could be unambiguously detected via C-D signature peaks in single-cell Raman spectra, and the obtained labeling pattern was confirmed by nanoscale-resolution secondary ion MS. In fast-growing Escherichia coli cells, label detection was already possible after 20 min. For functional analyses of microbial communities, the detection of D incorporation from D2O in individual microbial cells via Raman microspectroscopy can be directly combined with FISH for the identification of active microbes. Applying this approach to mouse cecal microbiota revealed that the host-compound foragers Akkermansia muciniphila and Bacteroides acidifaciens exhibited distinctive response patterns to amendments of mucin and sugars. By Raman-based cell sorting of active (deuterated) cells with optical tweezers and subsequent multiple displacement amplification and DNA sequencing, novel cecal microbes stimulated by mucin and/or glucosamine were identified, demonstrating the potential of the nondestructive D2O-Raman approach for targeted sorting of microbial cells with defined functional properties for single-cell genomics.
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Óxido de Deuterio/metabolismo , Consorcios Microbianos , Animales , Archaea/genética , Archaea/aislamiento & purificación , Archaea/metabolismo , Bacterias/genética , Bacterias/aislamiento & purificación , Bacterias/metabolismo , Secuencia de Bases , Biomasa , Ciego/microbiología , ADN de Archaea/genética , ADN Bacteriano/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Humanos , Hibridación Fluorescente in Situ , Ratones , Ratones Endogámicos C57BL , Consorcios Microbianos/genética , Técnicas Microbiológicas , Datos de Secuencia Molecular , Pinzas Ópticas , Filogenia , Espectrometría RamanRESUMEN
Methanogenic archaea in the bovine rumen are responsible for the reduction of carbon molecules to methane, using various electron donors and driving the electron flow across the microbial food webs. Thus, methanogens play a key role in sustaining rumen metabolism and function. Research of rumen methanogenic archaea typically focuses on their composition and function in mature animals, while studies of early colonization and functional establishment remain scarce. Here, we investigated the metabolic potential and taxonomic composition of the methanogenic communities across different rumen developmental stages. We discovered that the methanogenesis process changes with age and that the early methanogenic community is characterized by a high activity of methylotrophic methanogenesis, likely performed by members of the order Methanosarcinales, exclusively found in young rumen. In contrast, higher hydrogenotrophic activity was observed in the mature rumen, where a higher proportion of exclusively hydrogenotrophic taxa are found. These findings suggest that environmental filtering acts on the archaeal communities and select for different methanogenic lineages during different growth stages, affecting the functionality of this ecosystem. This study provides a better understanding of the compositional and metabolic changes that occur in the rumen microbiome from its initial stages of colonization and throughout the animals' life.
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Tracto Gastrointestinal/microbiología , Metano/metabolismo , Methanosarcinales/metabolismo , Microbiota , Rumen/microbiología , Animales , Bovinos , Hidrógeno/metabolismo , Methanosarcinales/clasificaciónRESUMEN
Dietary changes are known to affect gut community structure, but questions remain about the mechanisms by which diet induces shifts in microbiome membership. Here, we addressed these questions in the rumen microbiome ecosystem - a complex microbial community that resides in the upper digestive tract of ruminant animals and is responsible for the degradation of the ingested plant material. Our dietary intervention experiments revealed that diet affects the most abundant taxa within the microbiome and that a specific group of methanogenic archaea of the order Methanomicrobiales is highly sensitive to its changes. Using metabolomic analyses together with in vitro microbiology approaches and whole-genome sequencing of Methanomicrobium mobile, a key species within this group, we identified that redox potential changes with diet and is the main factor that causes these dietary induced alternations in this taxa's abundance. Our genomic analysis suggests that the redox potential effect stems from a reduced number of anti-reactive oxygen species proteins coded in this taxon's genome. Our study highlights redox potential as a pivotal factor that could serve as a sculpturing force of community assembly within anaerobic gut microbial communities.
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Archaea/aislamiento & purificación , Microbioma Gastrointestinal , Tracto Gastrointestinal/microbiología , Rumen/microbiología , Alimentación Animal/análisis , Animales , Archaea/clasificación , Archaea/genética , Bovinos , Dieta , Oxidación-ReducciónRESUMEN
The cellulosome is an extracellular multi-enzyme complex that is considered one of the most efficient plant cell wall-degrading strategies devised by nature. Its unique modular architecture, achieved by high affinity and specific interaction between protein modules (cohesins and dockerins) enables formation of various enzyme combinations. Extensive research has been dedicated to the mechanistic nature of the cellulosome complex. Nevertheless, little is known regarding its distribution and abundance among microbes in natural plant fibre-rich environments. Here, we explored these questions in bovine rumen microbial communities, specialized in efficient degradation of lignocellulosic plant material. We bioinformatically screened for cellulosomal modules in this complex environment using a previously published ultra-deep fibre-adherent rumen metagenome. Intriguingly, a large portion of the functions of the dockerin-containing proteins were related to alternative biological processes, and not necessarily to the classic fibre degradation function. Our analysis was experimentally validated by characterizing specific interactions between selected cohesins and dockerins and revealed that cellulosome is a more generalized strategy used by diverse bacteria, some of which were not previously associated with cellulosome production. Remarkably, our results provide additional proof of similarity among rumen microbial communities worldwide. This study suggests a broader and widespread role for the cellulosomal machinery in nature.
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Bacterias/aislamiento & purificación , Celulosomas/enzimología , Microbioma Gastrointestinal , Filogenia , Rumen/microbiología , Animales , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bovinos , Celulosomas/genética , Metagenoma , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismoRESUMEN
Ruminococcus champanellensis is a keystone species in the human gut that produces an intricate cellulosome system of various architectures. A variety of cellulosomal enzymes have been identified, which exhibit a range of hydrolytic activities on lignocellulosic substrates. We describe herein a unique R. champanellensis scaffoldin, ScaK, which is expressed during growth on cellobiose and comprises a cohesin module and a family 25 glycoside hydrolase (GH25). The GH25 is non-autolytic and exhibits lysozyme-mediated lytic activity against several bacterial species. Despite the narrow acidic pH curve, the enzyme is active along a temperature range from 2 to 85°C and is stable at very high temperatures for extended incubation periods. The ScaK cohesin was shown to bind selectively to the dockerin of a monovalent scaffoldin (ScaG), thus enabling formation of a cell-free cellulosome, whereby ScaG interacts with a divalent scaffodin (ScaA) that bears the enzymes either directly or through additional monovalent scaffoldins (ScaC and ScaD). The ScaK cohesin also interacts with the dockerin of a protein comprising multiple Fn3 domains that can potentially promote adhesion to carbohydrates and the bacterial cell surface. A cell-free cellulosomal GH25 lysozyme may provide a bacterial strategy to both hydrolyze lignocellulose and repel eventual food competitors and/or cheaters.
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Proteínas Bacterianas/metabolismo , Celulosomas/enzimología , Muramidasa/metabolismo , Ruminococcus/enzimología , Proteínas Bacterianas/genética , Membrana Celular/metabolismo , Celulosa/metabolismo , Celulosomas/genética , Celulosomas/metabolismo , Humanos , Muramidasa/genética , Ruminococcus/genética , Ruminococcus/metabolismoRESUMEN
Based on a previous ensiling study in glass silos of various pomegranate pulp (PP) mixtures, fresh pomegranate pulp (PP) was mixed with drier feeds including soy hulls and corn silage (40:35:25 on DM basis) and ensiled in 32 pressed bales (700 kg each) wrapped with stretch polyethylene film. This ensiled pomegranate pulp mixture (PPM) was included in lactating cow total mixed ration (TMR) at a level of 20% of DM (PPM-TMR). Performance and digestion experiment was conducted with two groups of 21 milking cows each, fed individually one of the two TMR: 1. Control TMR without ensiled PPM; 2. Experimental TMR which contained 20% ensiled PPM, including 8% PP as corn grain replacer. Voluntary DM intake of cows fed the control TMR was 5.04% higher than that of the PPM cows. In vivo digestibility of DM, OM, NDF, CP and fat were significantly higher in the control cows compared with the PPM group, but methane production in the rumen fluid was 25% lower in the PPM cows. A slightly higher milk yield (by 2.2%) observed in the control cows; however, milk fat content was 5.9% higher in the PPM cows. This was reflected in similar yield of energy corrected milk (ECM) and 3.97% increase in production efficiency (ECM/DM intake) of the PPM cows compared with the control ones. Welfare of the cows, as assessed by length of daily recumbence time, was in the normal range for both groups. Body weight gain was also similar in both groups. Data suggest that the level of 8% PP in the PPM-TMR used in this study was probably too high for lactating cows and should be lowered to 4% in order to achieve better performance.
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Bovinos/fisiología , Digestión/efectos de los fármacos , Lactancia/efectos de los fármacos , Lythraceae , Ensilaje/análisis , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Dieta/veterinaria , Digestión/fisiología , Femenino , Lactancia/fisiología , AguaRESUMEN
Plasmids are self-replicating genetic elements capable of mobilization between different hosts. Plasmids often serve as mediators of lateral gene transfer, a process considered to be a strong and sculpting evolutionary force in microbial environments. Our aim was to characterize the overall plasmid population in the environment of the bovine rumen, which houses a complex and dense microbiota that holds enormous significance for humans. We developed a procedure for the isolation of total rumen plasmid DNA, termed rumen plasmidome, and subjected it to deep sequencing using the Illumina paired-end protocol and analysis using public and custom-made bioinformatics tools. A large number of plasmidome contigs aligned with plasmids of rumen bacteria isolated from different locations and at various time points, suggesting that not only the bacterial taxa, but also their plasmids, are defined by the ecological niche. The bacterial phylum distribution of the plasmidome was different from that of the rumen bacterial taxa. Nevertheless, both shared a dominance of the phyla Firmicutes, Bacteroidetes, and Proteobacteria. Evidently, the rumen plasmidome is of a highly mosaic nature that can cross phyla. Interestingly, when we compared the functional profile of the rumen plasmidome to two plasmid databases and two recently published rumen metagenomes, it became apparent that the rumen plasmidome codes for functions, which are enriched in the rumen ecological niche and could confer advantages to their hosts, suggesting that the functional profiles of mobile genetic elements are associated with their environment, as has been previously implied for viruses.
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Plastidios , Rumen/microbiología , Animales , Bovinos , FilogeniaRESUMEN
Plasmids are pivotal in driving bacterial evolution through horizontal gene transfer. Here, we investigated 3467 human gut microbiome samples across continents and disease states, analyzing 11,086 plasmids. Our analyses reveal that plasmid dispersal is predominantly stochastic, indicating neutral processes as the primary driver of their wide distribution. We find that only 20-25% of plasmid DNA is being selected in various disease states, constraining its distribution across hosts. Selective pressures shape specific plasmid segments with distinct ecological functions, influenced by plasmid mobilization lifestyle, antibiotic usage, and inflammatory gut diseases. Notably, these elements are more commonly shared within groups of individuals with similar health conditions, such as Inflammatory Bowel Disease (IBD), regardless of geographic location across continents. These segments contain essential genes such as iron transport mechanisms- a distinctive gut signature of IBD that impacts the severity of inflammation. Our findings shed light on mechanisms driving plasmid dispersal and selection in the human gut, highlighting their role as carriers of vital gene pools impacting bacterial hosts and ecosystem dynamics.
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Ecosistema , Enfermedades Inflamatorias del Intestino , Humanos , Plásmidos/genética , Bacterias/genética , Antibacterianos , Transferencia de Gen Horizontal , Enfermedades Inflamatorias del Intestino/genéticaRESUMEN
Current views of O2 accumulation in Earth history depict three phases: The onset of O2 production by â¼2.4 billion years ago; 2 billion years of stasis at â¼1 % of modern atmospheric levels; and a rising phase, starting about 500 million years ago, in which oxygen eventually reached modern values. Purely geochemical mechanisms have been proposed to account for this tripartite time course of Earth oxygenation. In particular the second phase, the long period of stasis between the advent of O2 and the late rise to modern levels, has posed a puzzle. Proposed solutions involve Earth processes (geochemical, ecosystem, day length). Here we suggest that Earth oxygenation was not determined by geochemical processes. Rather it resulted from emergent biological innovations associated with photosynthesis and the activity of only three enzymes: 1) The oxygen evolving complex of cyanobacteria that makes O2; 2) Nitrogenase, with its inhibition by O2 causing two billion years of oxygen level stasis; 3) Cellulose synthase of land plants, which caused mass deposition and burial of carbon, thus removing an oxygen sink and therefore increasing atmospheric O2. These three enzymes are endogenously produced by, and contained within, cells that have the capacity for exponential growth. The catalytic properties of these three enzymes paved the path of Earth's atmospheric oxygenation, requiring no help from Earth other than the provision of water, CO2, salts, colonizable habitats, and sunlight.
RESUMEN
Cellulosomes are intricate cellulose-degrading multi-enzymatic complexes produced by anaerobic bacteria, which are valuable for bioenergy development and biotechnology. Cellulosome assembly relies on the selective interaction between cohesin modules in structural scaffolding proteins (scaffoldins) and dockerin modules in enzymes. Although the number of tandem cohesins in the scaffoldins is believed to determine the complexity of the cellulosomes, tandem dockerins also exist, albeit very rare, in some cellulosomal components whose assembly and functional roles are currently unclear. In this study, we characterized the structure and mode of assembly of a tandem bimodular double-dockerin, which is connected to a putative S8 protease in the cellulosome-producing bacterium, Clostridium thermocellum. Crystal and NMR structures of the double-dockerin revealed two typical type I dockerin folds with significant interactions between them. Interaction analysis by isothermal titration calorimetry and NMR titration experiments revealed that the double-dockerin displays a preference for binding to the cell-wall anchoring scaffoldin ScaD through the first dockerin with a canonical dual-binding mode, while the second dockerin module was unable to bind to any of the tested cohesins. Surprisingly, the double-dockerin showed a much higher affinity to a cohesin from the CipC scaffoldin of Clostridium cellulolyticum than to the resident cohesins from C. thermocellum. These results contribute valuable insights into the structure and assembly of the double-dockerin module, and provide the basis for further functional studies on multiple-dockerin modules and cellulosomal proteases, thus highlighting the complexity and diversity of cellulosomal components.